Trapping and Characterization of the Reaction Intermediate in Cyclodextrin Glycosyltransferase by Use of Activated Substrates and a Mutant Enzyme
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- 作者：Renee Mosi ; Shouming He ; Joost Uitdehaag ; Bauke W. Dijkstra ; and Stephen G. Withers
- 刊名：Biochemistry
- 出版年：1997
- 出版时间：August 12, 1997
- 年：1997
- 卷：36
- 期：32
- 页码：9927 - 9934
- 全文大小：307K
- 年卷期：v.36,no.32(August 12, 1997)
- ISSN：1520-4995
文摘
Cyclodextrin glycosyltransferases (CGTases) catalyze thedegradation of starch into linear orcyclic oligosaccharides via a glycosyl transfer reaction occurring withretention of anomeric configuration.They are also shown to catalyze the coupling ofmaltooligosaccharyl fluorides. Reaction is thought toproceed via a double-displacement mechanism involving a covalentglycosyl-enzyme intermediate. Thisintermediate can be trapped by use of 4-deoxymaltotriosyl 
-fluoride(4DG3F). This substrate containsa good leaving group, fluoride, thus facilitating formation of theintermediate, but cannot undergo thetransglycosylation step since the nucleophilic hydroxyl group at the4-position is missing. When 4DG3Fwas reacted with wild-type CGTase (Bacilluscirculans 251), it was found to be a slow substrate(kcat =2 s-1) compared with the parent glycosylfluoride, maltotriosyl -fluoride (kcat =275 s-1). Unfortunately,a competing hydrolysis reaction reduces the lifetime of theintermediate precluding its trapping andidentification. However, when 4DG3F was used in the presence ofthe presumed acid/base catalystmutant Glu257Gln, the intermediate could be trapped and analyzedbecause the first step remained fastwhile the second step was further slowed (kcat= 0.6 s-1). Two glycosylated peptideswere identified ina proteolytic digest of the inhibited enzyme by means of neutral losstandem mass spectrometry. Edmansequencing of these labeled peptides allowed identification of Asp229as the catalytic nucleophile andprovided evidence for a covalent intermediate in CGTase. Asp229 isfound to be conserved in all membersof the family 13 glycosyl transferases.
-fluoride(4DG3F). This substrate containsa good leaving group, fluoride, thus facilitating formation of theintermediate, but cannot undergo thetransglycosylation step since the nucleophilic hydroxyl group at the4-position is missing. When 4DG3Fwas reacted with wild-type CGTase (Bacilluscirculans 251), it was found to be a slow substrate(kcat =2 s-1) compared with the parent glycosylfluoride, maltotriosyl -fluoride (kcat =275 s-1). Unfortunately,a competing hydrolysis reaction reduces the lifetime of theintermediate precluding its trapping andidentification. However, when 4DG3F was used in the presence ofthe presumed acid/base catalystmutant Glu257Gln, the intermediate could be trapped and analyzedbecause the first step remained fastwhile the second step was further slowed (kcat= 0.6 s-1). Two glycosylated peptideswere identified ina proteolytic digest of the inhibited enzyme by means of neutral losstandem mass spectrometry. Edmansequencing of these labeled peptides allowed identification of Asp229as the catalytic nucleophile andprovided evidence for a covalent intermediate in CGTase. Asp229 isfound to be conserved in all membersof the family 13 glycosyl transferases.